The present invention relates generally to the examination of tissues using optical spectroscopy and relates more particularly to a method and an apparatus for examining a tissue using the spectral wing emission therefrom induced by visible to infrared photoexcitation.
Optical spectroscopy has received increasing attention over the past several years as a tool for use in examining tissues. One such application of optical spectroscopy to the examination of tissues has been in the detection of cancer and precancerous states and has involved the use of steady-state native fluorescence. For example, in U.S. Pat. No.4,930,516, inventors Alfano et al., which issued Jun. 5, 1990, and which is incorporated herein by reference, there is disclosed a method and apparatus for detecting the presence of cancerous tissue using visible luminescence. According to the aforementioned patent, the tissue to be examined is excited with a beam of monochromatic light that causes the tissue to fluoresce over a spectrum of wavelengths. The monochromatic light disclosed in the patent has a wavelength in the range of 350-500 nm. The intensity at which the excited tissue fluoresces is measured either over a spectrum or at a predetermined number of preselected wavelengths, such as at 531 nm, 522 nm and 633 nm. The patent further teaches that one can then determine the carcinomatoid status of the tissue in question by comparing the detected spectrum, one or more peak wavelengths of the detected spectrum, or a ratio or difference of particular wavelengths from the detected spectrum to standards obtained from known tissues.
Another example of the use of optical spectroscopy, particularly steady-state native fluorescence, in the detection of cancer and precancerous states is disclosed in U.S. Pat. No. 5,131,398, inventors Alfano et al., which issued Jul. 21, 1992, and which is incorporated herein by reference. In the aforementioned patent, there is disclosed a method and apparatus for distinguishing cancerous tumors and tissue from benign tumors and tissue or normal tissue using native fluorescence. According to one embodiment of said patent, the tissue to be examined is excited with a beam of monochromatic light at 300 nm. The intensity of the native fluorescence emitted from the tissue is measured at 340 nm and at 440 nm. The ratio of the two intensities is then calculated and used as a basis for determining if the tissue is cancerous as opposed to benign or normal. According to another embodiment of said patent, excitation profiles may be employed to distinguish cancerous tissue from benign or normal tissue. For example, the patent teaches that excitation spectra obtained by measuring the intensity of fluorescence at 340 nm as the excitation wavelength is varied from 220 nm to 325 nm are different for cancerous and benign breast tissues.
Other patents and publications that relate to the use of steady-state native fluorescence in the detection of cancer and precancerous states include the following: U.S. Pat. No. 5,042,494, inventor Alfano, issued Aug. 27, 1991; U.S. Pat. No. 5,413,108, inventor Alfano, issued May 9, 1995; U.S. Pat. No. 5,769,081, inventors Alfano et al., issued Jun. 23, 1998; U.S. Pat. No. 5,612,540, inventors Richards-Kortum et al., issued Mar. 18, 1997; U.S. Pat. No. 4,957,114, inventors Zeng et al., issued Sep. 18, 1990; Yang et al., xe2x80x9cFundamental Differences of Excitation Spectrum between Malignant and Benign Breast Tissues,xe2x80x9d Photochemistry and Photobiology, 66(4):518-22 (1997); Yang et al., xe2x80x9cExcitation Spectrum of Malignant and Benign Breast Tissues: A Potential Optical Biopsy Approach,xe2x80x9d Lasers in the Life Sciences, 7(4):249-65 (1997); Galeotti et al., xe2x80x9cOn the Fluorescence of NAD(P)H in Whole-Cell Preparations of Tumours and Normal Tissues,xe2x80x9d Eur. J. Biochem., 17:485-96 (1970); and Japanese Patent Application No. Sho-57-795, published Jul. 15, 1983, all of which are incorporated herein by reference.
In addition, it should be noted that steady-state native fluorescence has also been used to detect a number of other abnormal or disease states unrelated to cancer, such as the detection of caries in teeth (U.S. Pat. No. 4,479,499, inventor Alfano, which issued Oct. 30, 1984, and which is incorporated herein by reference) and the detection of atherosclerotic plaque in arteries (U.S. Pat. No. 4,913,142, inventors Kittrell et al., issued Apr. 3, 1990, and which is incorporated herein by reference).
Another type of technique for detecting cancer in tissues has involved the use of time-resolved fluorescence spectroscopy and is exemplified by U.S. Pat. No. 5,348,018, inventors Alfano et al., which issued Sep. 20, 1994, and U.S. Pat. No. 5,467,767, inventors Alfano et al., which issued Nov. 21, 1995, both of which are incorporated herein by reference. In, for example, the aforementioned U.S. Pat. No.5,348,018, there is disclosed a method for determining if tissue is malignant as opposed to non-malignant (i.e., benign tumor tissue, benign tissue, or normal tissue), said method comprising, in one embodiment, irradiating a human breast tissue sample with light at a wavelength of about 310 nm and measuring the time-resolved fluorescence emitted therefrom at about 340 nm. The time-resolved fluorescence profile is then compared to similar profiles obtained from known malignant and non-malignant human breast tissues. By fitting the profiles to the formula I(t)=A1e(xe2x88x92t/xcfx841)+A2e(xe2x88x92t/xcfx842), one can quantify the differences between tissues of various conditions. For example, non-malignant human breast tissues exhibit a slow component (xcfx842) which is less than 1.6 ns whereas malignant human breast tissues exhibit a slow component (xcfx842) which is greater than 1.6 ns. In addition, non-malignant human breast tissues exhibit a ratio of fast to slow amplitudes (A1/A2) which is greater than 0.85 whereas malignant human breast tissue exhibit a ratio of fast to slow amplitudes (A1/A2) which is less than 0.6. This technique can be used with different excitation and/or emission wavelengths, and can be applied to the detection of malignancies (or other abnormal states) in tissues other than human breast tissue.
It should be noted that conventional fluorescence spectroscopic techniques of the types described above for detecting cancerous or precancerous states in tissues, whether of the steady-state variety or of the time-resolved variety, have typically involved using photoexcitation wavelengths far below 600 nm, said photoexcitation wavelengths typically residing in the range of about 300 to 500 nm.
Another type of spectroscopic technique that has been used to examine tissues has involved the use of Raman spectroscopy. One such application of Raman spectroscopy to the examination of tissues has been in the detection of cancer and is exemplified by U.S. Pat. No.5,261,410, inventors Alfano et al., which issued Nov. 16, 1993, and which is incorporated herein by reference. In the aforementioned patent, there is disclosed a method for determining if a tissue is a malignant tumor tissue, a benign tumor tissue, or a normal or benign tissue. The method is based on the discovery that, when irradiated with a beam of infrared monochromatic light, malignant tumor tissue, benign tumor tissue, and normal or benign tissue produce distinguishable Raman spectra. For human breast tissue, some salient differences in the respective Raman spectra are the presence of four Raman bands at a Raman shift of about 1078, 1300, 1445 and 1651 cmxe2x88x921 for normal or benign tissue, the presence of three Raman bands at a Raman shift of about 1240, 1445 and 1659 cmxe2x88x921 for benign tumor tissue, and the presence of two Raman bands at a Raman shift of about 1445 and 1651 cmxe2x88x921 for malignant tumor tissue. In addition, it was discovered that for human breast tissue the ratio of intensities of the Raman bands at a Raman shift of about 1445 and 1659 cmxe2x88x921 is about 1.25 for normal or benign tissue, about 0.93 for benign tumor tissue, and about 0.87 for malignant tumor tissue.
In addition, as exemplified by U.S. Pat. No. 5,293,872, inventors Alfano et al., which issued Mar. 15, 1994, and which is incorporated herein by reference, Raman spectroscopy has also been used to distinguish between calcified atherosclerotic tissue and fibrous atherosclerotic tissue or normal cardiovascular tissue.
It should be noted that conventional Raman spectroscopic techniques of the types described above have typically involved using photoexcitation wavelengths in the range of about 680 nm to 1350 nm.
When using Raman spectroscopy to examine tissues, there is often detected, in addition to the desired Raman bands, an undesired fluorescence emission, which appears as background and is typically regarded as noise. Typically, the longer the photoexcitation wavelength, the smaller the fluorescence emission detected. See Frank et al., xe2x80x9cCharacterization of human breast specimens with Near-IR Raman spectroscopy,xe2x80x9d Anal. Chem., 66(3):319-26 (1994), which is incorporated herein by reference. More specifically, where the photoexcitation wavelength is in much of the visible portion of the spectrum, the fluorescence is generally so large from the tissue that the Raman signature lines are not visible. Where, however, the photoexcitation wavelength is longer, e.g., in the range of about 632 nm to 980 nm, the Raman lines are usually visible, but the fluorescence emission is typically stronger than the Raman emission. However, because the fluorescence emission in this situation is typically regarded as noise since it is the Raman emission that is of interest, complex fitting parameters have been devised to subtract that portion of the detected signal attributable to fluorescence emission in order to identify those spectral features associated with the Raman active vibration modes. See Baraga et al., xe2x80x9cRapid near-infrared Raman spectroscopy of human tissue with a spectrograph and CCD detector,xe2x80x9d Appl. Spectrosc., 46(2):187-90 (1992) and Mahadevan et al., xe2x80x9cOptical techniques for the diagnosis of cervical precancers: Comparison of Raman and fluorescence spectroscopies,xe2x80x9d in Advances in Fluorescence Sensing Technology II, J. R. Lakowicz, ed., Proc. SPIE, 2388:110-20 (1995), both of which are incorporated herein by reference.
Accordingly, in view of the above, it can be seen that far red and near infrared spectral wing (SW) emissions, where incidentally induced by photoexcitation of tissues with light having a wavelength of at least 600 nm, have not heretofore been used to detect cancerous tissues or other abnormal or diseased tissue states, such as diabetics, Alzheimers, sleep disorders, aging, lung disorders, blood flow, bile-ruben infection, bums, and SW emission wavelengths are at least 20 nm more than the excitation wavelength.
It is an object of the present invention to provide a new method for examining a tissue.
It is another object of the present invention to provide a new method for examining a tissue that involves the use of the spectral wing emission from the tissue induced by visible (in particular, visible having a wavelength greater than about 600 nm) to infrared photoexcitation of the tissue.
It is yet another object of the present invention to provide a method as described above that can be used for either in vivo or in vitro examination of a tissue.
Therefore, according to one aspect of the invention, there is provided a method for characterizing the condition of a tissue sample (i.e., characterizing the tissue sample as normal, cancerous, precancerous, adipose, etc.), said method comprising the steps of: (a) photoexciting the tissue sample with substantially monochromatic light having a wavelength of at least 600 nm; and (b) using the resultant far red and near infrared spectral wing emitted from the tissue sample to characterize the condition of the tissue sample for emission wavelength greater than 650 nm.
In one embodiment, the substantially monochromatic photoexciting light is a continuous beam of light and said using step comprises using the resultant steady-state native far red and near infrared spectral wing emitted from the tissue sample to characterize the condition of the tissue sample. Preferably, the photoexciting light has a wavelength in the range of about 600 to 980 nm. The SW emission wavelength is at least 20 nm greater than the excitation wavelength (for example, excite with 630 nm and measure SW from 650 nm to 950 nm). Said using step may comprise obtaining a spectral profile of the steady-state far red and near infrared spectral wing emitted from the tissue sample and comparing said spectral profile to standards obtained from tissues whose conditions are known. This may be done, for example, by determining the normalized integrated intensity of the spectral profile and comparing said normalized integrated intensity to appropriate standards or by determining a ratio or difference of intensities at two wavelengths along said spectral profile and comparing said ratio or difference to appropriate standards. Said using step may alternatively comprise detecting the resultant steady-state far red and near infared spectral wing emission at two wavelengths (instead of along a spectrum), determining a ratio or difference of intensities at said two wavelengths and comparing said ratio or difference to appropriate standards.
In another embodiment, the substantially monochromatic photoexciting light is a light pulse and said using step comprises using the resultant time-resolved far red and near infrared spectral wing emitted from the tissue sample to characterize the condition of the tissue sample. Preferably, the photoexciting light has a wavelength in the range of about 600 to 980 nm. The SW emission wavelength is at least 20 nm greater than excitation wavelength (for example, excite with 800 nm and SW emission wavelengths are from 840 nm to 950 nm). Said using step may comprise obtaining a profile of the time-resolved SW emitted from the tissue sample and comparing said profile to standards obtained from tissues whose conditions are known. This may be done, for example, by fitting the time-resolved profile to the formula I(t)=A1e(xe2x88x92t/xcfx841)+A2e(xe2x88x92t/xcfx842) and comparing the resultant values for A1/A2 and/or xcfx841 with appropriate standards.
In still,another embodiment, the substantially monochromatic photoexciting light is a polarized light pulse and said using step comprises using the parallel and perpendicular components of the resultant polarized time-resolved far red and near infrared spectral wing emitted from the tissue sample to characterize the condition of the tissue sample. Preferably, the photoexciting light has a wavelength in the range of about 600 to 980 nm. Said using step may comprise obtaining a profile of one or both of the parallel and perpendicular components of the polarized time-resolved far red and near infrared spectral wing emitted from the tissue sample and comparing said profile(s) to standards obtained from tissues whose conditions are known. This may be done, for example, by fitting the time-resolved profile(s) to the formula I(t)=A1e(xe2x88x92t/xcfx841)+A2e(xe2x88x92t/xcfx842) and comparing the resultant values with appropriate standards.
According to another aspect of the invention, there is provided a method for imaging a tissue sample, said method comprising the steps of: (a) photoexciting the tissue sample with substantially monochromatic light having a wavelength of at least 600 nm; and (b) using the resultant far red and near infrared spectral wing emitted from the tissue sample to form an image of the tissue sample from 650 nm to 950 nm.
It is even yet another object of the present invention to provide a new apparatus for imaging a tissue.
It is still yet another object of the present invention to provide a new apparatus for imaging a tissue that involves the use of the spectral wing emission from the tissue induced by far red (in particular, visible having a wavelength greater than about 600 nm) to infrared photoexcitation of the tissue.
It is a further object of the present invention to provide an apparatus as described above that can be used for the in vivo or in vitro imaging of a tissue.
Therefore, according to another aspect of the invention, there is provided an apparatus for imaging a tissue sample, said apparatus comprising: (a) a light source for photoexciting the tissue sample with substantially monochromatic light having a wavelength of at least 600 nm; (b) a light detector; (c) collection optics for imaging the light emitted from the tissue sample from 650 nm to 950 nm onto the light detector; (d) filter means positioned between the tissue sample and the light detector for selectively transmitting, from the light emitted from the tissue sample, light having a wavelength greater than said substantially monochromatic light by at least 20 nm; and (e) a display coupled to said light detector for displaying an image of the tissue sample based on the light detected by said light detector.
Preferably, said apparatus is further provided with the instrumentalities needed to perform one or more of the techniques described above for characterizing the condition of a tissue. In this manner, one can both image a tissue and indicate (for example, by a scheme of shading or coloring) the condition of the tissue.
Additional objects, as well as features, advantages and aspects of the present invention, will be set forth in part in the description which follows, and in part will be obvious from the description or may be learned by practice of the invention. In the description, reference is made to the accompanying drawings which form a part thereof and in which is shown by way of illustration specific embodiments for practicing the invention. These embodiments will be described in sufficient detail to enable those skilled in the art to practice the invention, and it is to be understood that other embodiments may be utilized and that structural changes may be made without departing from the scope of the invention. The following detailed description is, therefore, not to be taken in a limiting sense, and the scope of the present invention is best defined by the appended claims.